Student Name: Sam Arsenault, University of Georgia
Mentor Names: Zhaojie Deng, Dr. Leidong Mao, Dr. Jonathan Arnold, University of Georgia
By setting the cell’s internal “clock”, circadian rhythms are central to many cellular activities such as carbon metabolism and the cell cycle. Therefore, a detailed understanding of these rhythms is crucial for determining how to maximize the effectiveness of drugs and treatments for a variety of diseases. However, traditional methods for studying circadian rhythm, such as race tube analysis and fluorescent labeling, are only capable of observing the overarching rhythm in a large population of cells. They lack the resolution required to observe the clock in individual cells. We need to develop a new technique for tracking the activity of individual cells that has both versatility and accuracy. Progress in the field of microfluidics has led to a new method for isolating individual cells into droplets so that they can observed without interference from other cells. Using a PDMS droplet microfluidics device, we were able to gather data on the circadian rhythms of hundreds of isolated single cells. Preliminary data indicates that single cells do indeed have an intrinsic rhythm but it tends to be very noisy and stochastic. When the circadian rhythms of small (2-3 cell) clusters were observed, there was a noticeable decrease in the amount of noise in the system. Consequently, it was easier to see the circadian rhythms in these small clusters of cells. This leads us to the conclusion that a cellular communication mechanism may help to explain how stochastic and damping behaviors, common to single cell circadian rhythms, can be sidestepped so that cells synchronize en masse. Further investigation into this communication mechanism could yield crucial insight into how cells are able to work together synchronously at the organismal level.
Student Name: Jessica Bramhall, Virginia Tech
Autografts, allografts and joint replacements are the current methods used for repairing damaged bone and cartilage in joints. These methods are extensive procedures that cause a lot of pain as well as many post-operative limitations such as the inability to do certain physical activities. Using the technology of a 3D printer and stem cell culturing it is believed that we can print and grow healthy new bone and cartilage to repair damaged joints. Through the use of the AutoCAD software, we have designed a scaffold that meets the desired size restraints and successfully prints on a Solidoodle 3D printer. This scaffold will be printed and used for strength and stress testing, cell bondage testing, and tissue growth testing. Upon completion of these tests we will be able to determine if this method of bone and cartilage regeneration is a viable option for patients with degenerative joint conditions.
Student Name: Sina Mostafavi, University of North Carolina at Charlotte
It is widely accepted that cells grown in three-dimension (3D) culture more accurately mimic in vivo microenvironment. Numerous kinds of three-dimension culture methods have been reported with a wide range of physical, chemical and spatio properties. These culture methods mimic the in vivo microenvironment to a certain extent; however, for most of the 3D cultures, it is hard to state how close to the in vivo condition they are. Both academia and industry call for a standard biomarker for three dimensionality. A long-term goal in our laboratory is to explore the question of existence of biomarkers for different tissue type. As a first step, we are exploring the functional and structural characteristic indicator of three-dimensionality in nerve-derived micro tissue. This knowledge is a pre-requisite in early three-dimensionality biomarkers discovery experiment action. In this study, we are using a human neuroblastoma cell line (SHSY-5Y). Our hypothesis is that three-dimensionality in nerve tissue is characterized by low cytosolic calcium oscillation/spike frequency (functional) and high caveolae density (structural) in comparison to 2D cultures. We use the term “complex physiological \ relevance” (CPR) to describe such functional/ structural features that are exhibited only in three-dimension culture systems, but absent in its 2D counterpart.
Student Name: Christopher M. Jabczynski, University of Arizona
Rapid and precise methods to ascertain the presence of viruses are needed for clinical and industrial applications. Available methods are currently slow, imprecise, and have limited sensitivity. A silver (Ag) coated nanorod (AgNR) array in combination with surface enhanced raman spectroscopy (SERS) have been developed to rapidly detect viruses (30-60 seconds) with enhanced sensitivity and specificity. The AgNR substrate enables unimpeded molecular and structural characterization of viruses. This method provides high sensitivity, in the attomolar region, and exhibits the ability to detect trace levels of virus in material while forgoing manipulation or amplification of the virus. This research shows that SERS may detect molecular signatures from important respiratory RNA and DNA viruses. The viruses studied were Swine Influenza (SIV) and Porcine Respiratory and Reproductive Syndrome (PRRS), in pig oral samples, due to their impact on the pork industry and as a vector for transmission to humans. The AgNR substrate was made using oblique angle deposition (OAD) of Ag and Titanium (Ti) onto a glass backing, and polydimethylsiloxane (PDMS) was used to mold wells to hold the viruses on the substrate. Results were analyzed using principle component analysis. Polymerase chain reaction was used to confirm the presence of the viruses in the oral samples. The results show that SERS is able to provide real-time, direct, and rapid detection of viruses providing a link between the limited sensitivity of the available bioassays and the need for more rapid and sensitive detection of infectious viral agents.
Student Name: Pablo Diaz, University of Puerto Rico, Mayagüez Campus
Folding of the cortex is a critical process for the brain development, but the mechanisms are not completely understood. The main target of this project is to develop models and simulations of the brain’s cortical folding. We made a hypothesis in which differential growth combined with axonal tension can drive the folding process. Finite element modeling method in order to simulate the instability and bifurcation of cortical folding has been used. Results demonstrated that only one mechanism cannot explain the whole folding process. Differential growth has higher effect in the folding phenomenon, and skull constraint and fibers play regulator roles in morphological evolution of cortical folding. Finding a real model that covers the whole cortical folding process can give us an idea of how diseases during gestation cause abnormalities in the cortical folding, which may lead to mental disorders such as autism and severe retardation.
Student Name: Austin Schlirf, Clemson University
Fluorescence imaging is becoming an important tool in biomarker-guided diagnosis, staging, typing, and prognosis of cancer. However, in vivo fluorescence imaging suffers from suboptimal signal-to-noise ratio and shallow detection depth, caused by the strong tissue autofluorescence under external excitation and by the scattering and absorption of short-wavelength light in tissues. In this project, we tackle these limitations by using a novel type of optical nanoprobe, Zn3Ga2Ge2O10:Cr3+ (ZGGO:Cr) nanoparticles with very-long-lasting near-infrared (NIR) persistent luminescence. This allows optical imaging to be performed in an excitation-free and hence autofluorescence-free manner. The ZGGO:Cr nanoparticles were fabricated by a solvothermal method, followed by calcination at high temperature and wet grinding. ZGGO:Cr nanoparticles pre-charged by ultraviolet light exhibited NIR persistent luminescence (emission peaking at 696 nm and 713 nm) in the first biological transparency window (650–950 nm). Our studies reveal promising potential of these ZGGO:Cr nanoparticles as nanoprobes to detect chemical or biological changes, especially in the applications of cell tracking and tumor targeting.
Student: CJ Norsigian, University of Virginia
Chronic, incurable ocular diseases, such as, glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa require lifelong drug management to prevent blindness. These diseases affect millions of adults worldwide. We aim to design an implantable ocular drug delivery device consisting of microchannels embedded between top and bottom covers with a drug reservoir made from polydimethylsiloxane (PDMS). This type of device can be tailored to specific drugs that have been developed to treat the aforementioned diseases by adjusting the channel design to achieve the appropriate diffusion rate. We designed and fabricated two channel designs based around the corticosteroid Fluocinolone Acetonide. We based the channel design off the biological structures found in lymph nodes in an effort to create a contextualized design. Design generation, channel fabrication, and fluid path analysis are presented.
Student Name: Seyed R. Taghavi, University of Arkansas
Mycoplasma pneumonia is a major cause of respiratory disease in humans and accounts for as much as 20% of all community-acquired pneumonia. Existing mycoplasma diagnosis is primarily limited by the poor success rate at culturing the bacteria from clinical samples. There is a critical need to develop a new platform for mycoplasma detection that has high sensitivity, specificity, and expediency. Here we report the layer-by-layer (LBL) encapsulation of M. pneumoniae and mycoplasma commensal cells with Ag nanoparticles in two different polyelectrolyte matrixes; one matrix is the combination poly(allylamine hydrochloride) (PAH) with poly(styrene sulfonate) (PSS) and the other is poly(diallyldimethylammonium chloride) (PDADMAC) with PSS. We evaluated nanoparticle encapsulated mycoplasma cells as a platform for the differentiation of M. pneumoniae from mycoplasma commensal strains using surface enhanced Raman scattering (SERS) combined with multivariate statistical analysis. Pathogenic strains such as M. pneumoniae (M129) and M. genitalium, along with a series of commensal mycoplasma strains, were studied. Scanning electron microscopy, fluorescence imaging, and AFM showed that the Ag nanoparticles were incorporated between the oppositely charged polyelectrolyte layers. SERS spectra showed that LBL encapsulation provides excellent spectral reproducibility. Multivariate statistical analysis of the Raman spectra differentiated the pathogenic strains from the commensal strains with near 90-100% specificity and sensitivity, and low root mean square error. The technique shows promise for adaptation to sample preparation of M. pneumoniae infections in clinical specimens and represents a valuable alternative to current bacterial diagnostic techniques.
Student Name: Alyssa Huntington, Virginia Tech
Surface-bound polymers patterned with reactive functional groups provide means for cellular interaction studies. These surfaces are generated through the covalent attachment of a polymer, and then postpolymerization modification to produce a pattern using microcapillary printing. While this technique has been optimized in organic conditions, aqueous solutions may be necessary for use in biological settings. Aqueous solutions pose a risk when generating functional surfaces due to the possibility of hydrolysis. This study was performed to learn the limitations that biological settings pose on the generation of functionalized surfaces, including the necessity of triethylamine and the effect of pH on hydrolysis. Using FTIR, ellipsometry, and UV-vis analysis, it was found that triethylamine acts as a catalyst during functionalization. Hydrolysis resulting from different pH values in pure aqueous solutions and aqueous solutions containing DMF was measured. Hydrolysis of varying degrees occurred on all substrates exposed to these solutions, and ongoing investigations are being conducted to better characterize this relationship.
Student Name: Heather Bomberger, Virginia Tech
Cilia and flagella are cellular extensions that function in motility and sensing. Human sperm cells use cilia for locomotion and the multiple cilia of epithelial cells in the airways move foreign particles outward. Non-motile cilia in the eyes and nose function in the perception of light and chemical signals. Because cilia lack ribosomes, the RNA-protein particles responsible for protein synthesis, all proteins needed for cilia function or growth must be transported from the cell body into the protruding organelle. Intraflagellar transport (IFT) is one pathway for proteins transport from the base of the cilia, to the tip, and back again. Important steps of cilia assembly are thought to happen at the ciliary tip; cilia grow by the addition of subunits at the tip and IFT complexes are remodeled at the tip to allow their return to the cell body. The goal of my work is to develop an improved technique to image individual protein particles at the ciliary tip. Proteins of the IFT particles are made visible under the microscope via florescent protein (i.e. GFP) tagging. However, crowding of the particles at the ciliary tip largely obscures the visibility of individual proteins. A focused laser beam is used to bleach the fluorescence of some IFT particles at the tip. This increases the clarity for the remaining particles, but the tip is quickly refilled with florescent particles. We used a laser gate to control the bleaching laser; the laser blinks on and off at the base of the cilium in a pattern that will bleach most IFT particles but allows a few particles to pass unbleached through the gate, enter the cilium, and be imaged as individual particles while they complete their journey through the cilium. Further, we controlled the camera to not record while the bleaching laser is on; this will facilitate data analysis as it avoids recording of overexposed frames. This concept is carried out using a program written in Micro Manager and is communicated through an Arduino to the laser shutter. This program controls the laser shutter and the camera to capture movies of single IFT particles and their cargoes inside the cilia.